Degeneration of retinal ganglion cells (RGCs) and atrophy of their axons in the optic nerve are hallmarks of many ocular diseases and result in permanent vision loss. There is a critical need for new therapeutic strategies that induce robust axonal regeneration. The canonical Wnt/?3-catenin (?Wnt?) signaling pathway is one of several different Wnt pathways that regulate axon growth in the developing CNS. Although canonical Wnt ligands are known to be important for developmental axonal growth and neuronal viability throughout the CNS, their role in RGC axonal growth in adults after optic nerve injury is unknown. We recently demonstrated the novel finding that a single intravitreal injection of recombinant canonical Wnt3a ligand led to significant axon regeneration after murine optic nerve crush injury. In this project, we will ask whether repurposing the developmental axonal growth properties of canonical Wnt ligands can be used as a potential therapeutic strategy for axonal regeneration after optic nerve injury. Additionally, we will investigate how non-neuronal cells in the retina contribute to optic nerve regeneration and whether they modulate the activity of pro-regenerative factors. We demonstrated using a Wnt reporter transgenic mouse that Wnt3a-induced optic nerve regeneration was associated with upregulated Wnt signaling in the Muller glia and RGC, suggesting that Wnt promotes axonal regeneration by acting intrinsically within RGCs as well as extrinsically in non-RGCs such as Muller glia. Our central hypothesis is that activation of Wnt3a signaling within the retina leads to axonal regeneration, and that Wnt3a-induced axonal growth requires the involvement of Wnt signaling within both RGC and Muller glia. In Aim 1, we will build upon our promising preliminary data and will test the extent and duration of Wnt3a-induced axon regeneration after optic nerve crush in transgenic Wnt reporter mice at the functional and morphological levels, will determine the amount and localization of activated Wnt signaling and will identify underlying molecular mechanisms. In Aim 2, we will identify cellular mechanisms of axonal growth by determining the contribution of RGCs and Muller glia to Wnt- induced regeneration using cell-specific Wnt inhibitor and activator constructs. The collaborative team of the PI, an expert on Wnt and Muller glia, Dr. Kevin Park, an expert on optic nerve regeneration, and Drs. Ivanov and Porciatti, experts on RGCs, is uniquely suited to perform this study. This study will have a high impact on the field by defining a novel pro-regenerative activity for Wnt3a on the optic nerve, which will enhance the overall understanding of regeneration pathways and will identify novel targets for RGC regrowth. These findings could be applied to developing new therapies for regenerating damaged optic nerves, including traumatic injury, optic neuropathies, retinal ischemia and late stage glaucoma.